Diagnostic skin test for tuberculosis

ABSTRACT

Diagnostic methods capable of discriminating between cell mediated immunologic responses due to on the one hand active tuberculosis caused by bacteria belonging to the tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacterium bovis) and on the other hand vaccination with an immunogenic agent conferring immunity to tuberculosis. A diagnostic kit is also provided, comprising a polypeptide (e.g. MPT64) capable of eliciting a delayed type hypersensitivity reaction (Dth) in animals with active tuberculosis, but not in animals vaccinated against TB with an immunogenic agent (e.g. M. bovis BCG strain: Danish 1331). Also provided are polypeptide fragments comprising a T-cell epitope of MPT64 as well as nucleic acid fragments encoding these polypeptide fragments.

The present invention relates to a kit comprising as one part of the kita vaccine containing as the effective component an immunogenic agent(e.g. mycobacteria from the BCG strain: Danish 1331) capable ofconferring substantially increased immunity to tuberculosis, and as theother part of the kit at least one diagnostic skin test comprising apharmaceutical composition containing a polypeptide with which lymphoidcells previously primed with mycobacteria belonging to thetuberculosis-complex are capable of reacting and with which lymphoidcells previously primed with the immunogenic agent are not capable ofreacting, or a variant which is immunologically equivalent to thepolypeptide, as well as a method of diagnosing tuberculosis caused byMycobacterium tuberculosis, Mycobacterium africanum or Mycobacteriumbovis in a person, comprising intradermally injecting, in the person,the skin test, a positive skin response at the location of injectionbeing indicative of the person having or having had tuberculosis, and anegative skin response at the location of injection being indicative ofthe person not having or not having had tuberculosis, the polypeptidepreferably being MPT64 or an immunologically equivalent variant,analogue or subsequence thereof. The invention further relates to apharmaceutical composition comprising the polypeptide, a DNA fragmentencoding a polypeptide which is an immunological equivalent to MPT64,the polypeptide which is an immunological equivalent to MPT64, as wellas a method for vaccinating one or more persons in a population andsubsequently subjecting the population to a diagnostic test fortuberculosis by the method described above.

BACKGROUND

Tuberculosis remains a major world health problem. In fact, theincidence is increasing in both the so-called developing part of theworld as well as in industrialized countries like the United States ofAmerica. Recently, tuberculosis was ranked by the World HealthOrganization as the most frequent cause of death ascribable to a singleinfectious agent (Memorandum from a WHO meeting: Tuberculosis controland research strategies for the 1990s. Bulletin of the World HealthOrganization 70:17-21, 1992).

The means to effectively intervene transmission and thereby ultimatelyto get the disease under control are based on early diagnosis andtreatment combined with vaccination of the populations at risk. Thecurrently available anti-tuberculosis vaccine was developed in thebeginning of this century by Calmette and Guerin and is often referredto as "the Bacille Calmette et Guerin (BCG)". The vaccine strain evolvedafter serial passages of a virulent isolate of M. bovis on a bilecontaining growth medium. The resultant strain appeared to be avirulentfor humans. The nature of the loss of virulence is still not clearlyunderstood at the molecular level. However, the BCG vaccine is estimatedto be the most widely used live vaccine in the world and the remarkablelow number of serious complications observed as a consequence of the useof BCG clearly demonstrate that the strain is fully attenuated (Lotte etal., Adv. Tuberc. Res. 21, 107-193 (1984)). When the reports of thefirst successful vaccinations were published, several laboratories andvaccine producers around the world requested the strain from Calmetteand Guerin and the strain was subcultured locally under conditions whichvaried from one laboratory to another. This is the historical backgroundfor the occurrence of several substrains of BCG. Modern BCG producersmake use of freeze-lot systems which ensure that the genetic compositionof the bacteria--the product--has been conserved. Despite the widelyaccepted use of the BCG vaccine in many countries some countries neverintroduced it for use in general population vaccination programmes. Thisis the case in e.g. USA and Belgium. One of the reasons for thesecountries to be reluctant is that vaccination with BCG interferes withthe use of tuberculin skin testings for diagnosing tuberculosis and foruse in population surveys.

Infection of humans or susceptible animals with M. tuberculosis (orvaccination with BCG) will lead to the activation of the cellular branchof the immune system. The immunological status of a person (or animal)may therefore be monitored by analyses designed to measure the level oflymphoid cells primed against mycobacterial antigens. This may be donein vivo by measuring the "delayed type hypersensitivity (Dth) reaction"occurring 24 to 96 hours after the intracutaneous injection ofmycobacterial antigen.

The product which is currently used for elicitation of Dth reactions istuberculin--purified protein derivative (PPD). PPD consist of a crudemixture of proteins from M. tuberculosis. The proteins are recoveredfrom synthetic medium which has supported growth of the bacteria from 5to 6 weeks. The proteins are recovered by either ammonium sulphate ortrichloric acetic acid precipitation after heat inactivation and removalof the bacterial bodies from the cultures. Contaminating lipids may beremoved by ether extraction and low molecular components (<10,000) areremoved by ultrafiltration. However, the structural composition ofvirulent mycobacteria belonging to the tuberculosis complex (i.e. M.tuberculosis, M. bovis, and M. africanum) and the attenuated BCG strainis so closely related that the currently available PPD, due tocross-reactivity, will elicit a positive reaction in a large fraction ofthe vaccinated population. PPD is not a species specific reagent andpositive reactions may also be observed when people have been exposed toor infected with other mycobacterial species.

However, other reagents have been suggested as possible reagents in askin test for diagnosing tuberculosis. From WO 92/21697 a diagnosticskin test which comprises a 38 kDa lipoprotein or a 19 kDa fromMycobacterium tuberculosis is known. The skin test has specificity forMycobacterium tuberculosis infections, however, the skin test cannotdistinguish between patients immunised with BCG and patients sufferingfrom tuberculosis.

Furthermore, several mycobacterial proteins, e.g. MPT70 and MPT80 fromMycobacterium tuberculosis, as well as MPB64 from Mycobacterium bovishave been shown to elicit a Dth reaction in guinea pigs sensitized withmycobacteria belonging to the tuberculosis-complex. The gene encodingMPB64 has been cloned and sequenced (Yamaguchi et al. 1989) from M.bovis BCG Tokyo.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the invention to provide a kit comprising a vaccinefor immunizing a person against tuberculosis and furthermore comprisinga diagnostic skin test, whereby the immune status of the person withrespect to tuberculosis may be assessed before the vaccination orperiodically after the vaccination, the latter without having a positiveresponse in the diagnostic test due to the immunization caused by thevaccination itself.

Accordingly, the present invention relates to a kit for sequential usecomprising as one part of the kit a vaccine for immunizing a personagainst tuberculosis caused by Mycobacterium tuberculosis, Mycobacteriumafricanum or Mycobacterium bovis (=the tuberculosis complex), thevaccine comprising as the effective component an amount of animmunogenic agent effective in conferring substantial immunity totuberculosis, and as the other part of the kit at least one diagnosticskin test comprising a pharmaceutical composition containing apolypeptide with which lymphoid cells previously primed withmycobacteria belonging to the tuberculosis-complex are capable ofreacting and with which lymphoid cells previously primed with theimmunogenic agent are not capable of reacting, or a variant which isimmunologically equivalent to the polypeptide.

By the term "immunogenic agent" is meant any substance, composition ofmatter, or composition of organic material as for example a suspensionof cells or cell components, the immunogenic agent being capable ofconferring substantial immunity to tuberculosis in an animal, e.g. ahuman being, when administered in a suitable concentration/amount and inadmixture with suitable substances.

It will be understood that according to the invention T-cells primedwith this immunogenic agent should not react with a polypeptide capableof reacting with T-cells primed with mycobacteria belonging to thetuberculosis complex. The existence of such pairs of an immunogenicagent and a polypeptide has for the first time been demonstrated by theinventors and makes possible the distinction between vaccinatedindividuals and individuals with active tuberculosis.

An example of an immunogenic agent the properties described above isMycobacterium bovis BCG Copenhagen, from the Copenhagen BCG Laboratory,Statens Seruminstitut, Denmark, as described in table 2, example 5; thisBCG strain is hereinafter designated "BCG strain: Danish 1331". Togetherwith the protein MPT64, BCG strain: Danish 1331 forms such a pair.However, it is highly likely that other mycobacterial strains orcompositions comprised of components derived from mycobacteria willexhibit substantially the same immunological properties as BCG strain:Danish 1331, and such strains or compositions are also a part of the kitaccording to the invention.

Examples of strains which are likely to share the properties of M. bovisBCG strain: Danish 1331 are M. bovis BCG Glaxo, M. bovis BCG Pasteur, M.bovis BCG Canadian, and M. bovis BCG Tice. This is apparent from theresults in example 5, where it was found that the gene encoding MPT64apparently was lacking in these strains.

However, according to all aspects of the invention, BCG strain: Danish1331 is especially preferred as the immunogenic agent employed.

By stating that an agent "confers substantial immunity to tuberculosis"is meant that the vaccination of a person with a vaccine comprising theagent results in a substantially increased resistance to diseases causedby infections with bacteria belonging to the tuberculosis complex.Examples of such vaccines are well-known in the art, and a vaccinecontaining BCG strain: Danish 1331 exhibits these properties as doesvaccines containing other BCG strains.

By the term "polypeptide" is herein meant short peptides with a lengthof at least two amino acid residues and at most 10 amino acid residues,oligopeptides (11-100 amino acid residues), and longer peptides (theusual interpretation of "polypeptide", i.e. more than 100 amino acidresidues in length) as well as proteins (the functional entitycomprising at least one peptide, oligopeptide, or polypeptide which maybe chemically modified by being glycosylated, by being lipidated, or bycomprising prosthetic groups). The definition of polypeptides alsocomprises native forms of peptides/proteins in mycobacteria as well asrecombinant proteins or peptides in any type of expression vectorstransforming any kind of host, and also chemically synthesized peptides.

Furthermore, another object of the invention is a method of diagnosingtuberculosis, the method not resulting in positive responses in personspreviously vaccinated against tuberculosis but not actively infectedwith mycobacteria belonging to the tuberculosis complex.

Accordingly, another aspect of the invention is a method of diagnosingtuberculosis (active or previous) caused by Mycobacterium tuberculosis,Mycobacterium africanum or Mycobacterium bovis in a person, comprisingintradermally injecting, in the person, a pharmaceutical compositioncontaining a polypeptide with which lymphoid cells previously primedwith mycobacteria belonging to the tuberculosis complex are capable ofreacting and with which lymphoid cells previously primed with theabove-discussed immunogenic agent are not capable of reacting, or avariant which is immunologically equivalent to the polypeptide, apositive skin response at the location of injection being indicative ofthe person having or having had tuberculosis, and a negative skinresponse at the location of injection being indicative of the person nothaving or not having had tuberculosis.

In another aspect the present invention relates to a pharmaceuticalcomposition for diagnosing tuberculosis containing a polypeptide withwhich lymphoid cells previously primed with mycobacteria belonging tothe tuberculosis complex are capable of reacting and with which lymphoidcells previously primed with an immunogenic agent as discussed above arenot capable of reacting, or a variant of the polypeptide which isimmunologically equivalent to the polypeptide.

A further aspect of the invention is a DNA fragment comprising asubsequence or an analogue or a variant of the nucleotide sequence shownin SEQ ID NO: 1 (also shown in FIG. 1), the subsequence, analogue orvariant encoding a polypeptide which is immunologically equivalent tothe polypeptide encoded by the DNA sequence shown in SEQ ID NO: 1.

By the terms "analogue" or "variant" with regard to the DNA fragments ofthe invention is intended to indicate a nucleotide sequence whichencodes a polypeptide exhibiting identical or substantially identicalimmunological properties to a polypeptide encoded by a DNA fragment ofthe invention shown in SEQ ID NO: 1.

It is well known that the same amino acid may be encoded by variouscodes, the codon usage being related, inter alia, to the preference ofthe organisms in question expressing the nucleotide sequence. Thus, oneor more nucleotides or codons of a DNA fragment of the invention may beexchanged by others which, when expressed, result in a polypeptideidentical or substantially identical to the polypeptide encoded by theDNA fragment in question.

Therefore, the terms "analogue" and "variant" are used in the presentcontext to indicate a DNA fragment or a DNA sequence of a similarnucleotide composition or sequence as the DNA sequence encoding theamino acid sequence constituting MPT64, allowing for minor variationswhich do not have an adverse effect on the ligand binding propertiesand/or biological function and/or immunogenicity as compared to MPT64,or which give interesting and useful novel binding properties orbiological functions and immunogenicities etc. of the analogue. Theanalogous DNA fragment or DNA sequence may be derived from an animal ora human or may be partially or completely of synthetic origin asdescribed above. The analogue may also be derived through the use ofrecombinant DNA techniques.

Furthermore, the terms "analogue" and "subsequence" are intended toallow for variations in the sequence such as substitution, insertion(including introns), addition, deletion and rearrangement of one or morenucleotides, which variations do not have any substantial effect on thepolypeptide encoded by a DNA fragment or a subsequence thereof. The term"substitution" is intended to mean the replacement of one or morenucleotides in the full nucleotide sequence with one or more differentnucleotides, "addition" is understood to mean the addition of one ormore nucleotides at either end of the full nucleotide sequence,"insertion" is intended to mean the introduction of one or morenucleotides within the full nucleotide sequence, "deletion" is intendedto indicate that one or more nucleotides have been deleted from the fullnucleotide sequence whether at either end of the sequence or at anysuitable point within it, and "rearrangement" is intended to mean thattwo or more nucleotide residues have been exchanged with each other.

The variant or analogue of the DNA fragment in SEQ ID NO: 1 ispreferably one that hybridizes under stringent hybridization conditionswhich are to be understood in their conventional meaning, i.e. thathybridization is carried out at 65° C. in 2×SSC and final washing at 65°C. in 1×SSC using the method specified in the "Preamble" part of theExamples below.

In yet another aspect, the invention relates to a polypeptide having anamino acid sequence comprising a subsequence, an analogue or a variantof the amino acid sequence shown in SEQ ID NO: 2, the polypeptide beingimmunologically equivalent to the polypeptide having the amino acidsequence shown in SEQ ID NO: 2.

By the terms "analogue", "variant" and "subsequence" when used inconnection with polypeptides is meant any polypeptide having the sameimmunological characteristics as MPT64 with respect to being capable ofdiscriminating between infection with mycobacteria of the tuberculosiscomplex and vaccination with BCG strain: Danish 1331. Thus, included isalso a polypeptide from different sources, such as other bacteria oreven from eukaryotic cells.

The terms "analogue" and "variant" with regard to a polypeptide are alsoused in the present context to indicate a protein or polypeptide of asimilar amino acid composition or sequence as the characteristic aminoacid sequence shown in SEQ ID NO: 2, allowing for minor variations whichdo not have an adverse effect on the ligand binding properties and/orbiological function and/or immunogenicity, or which may give usefulnovel binding properties or biological functions and immunogenicitiesetc. of the analogue. The analogous polypeptide or protein may bederived from other microorganisms and the analogue may also be derivedthrough the use of recombinant DNA techniques.

As can be seen from the examples, a subsequence of the gene encodingMPT64 has been identified, a subsequence which most likely encodes aT-cell epitope responsible for the elicitation of the immunologicalresponse which can be read in a skin test. Thus, the polypeptide encodedby this DNA fragment (SEQ ID NO: 2, amino acids nos. 186-215) as well aspolypeptides encoded by analogues and subsequences of this DNA fragmentare preferred subsequences of the proteins of the invention, as areanalogues and variants of the polypeptide subsequence. Of course, alsothe DNA fragment (SEQ ID NO: 1, nucleotides nos. 694-783) as well asanalogues and subsequences encoding this polypeptide subsequence arepreferred DNA fragments of the invention. Especially interesting are DNAfragments of the invention comprising multiple copies of the DNAfragment encoding the T-cell epitope as are polypeptides of theinvention comprising multiple copies of T-cell epitopes, as these aresuspected to possess superior immunological properties over singleepitope variants of the polypeptide.

In the present context the term "immunologically equivalent" means thatthe polypeptide is functionally equivalent to the polypeptide having theamino acid sequence shown in SEQ ID NO: 2 with respect to its ability ofeliciting a Dth reaction to an extent of at least 45% of the Dthreaction elicited by the polypeptide under the same conditions, such asat least 65%, more preferred 85%, measured as the diameter of the Dthreaction.

In yet another aspect, the invention relates to a replicable expressionvector comprising the DNA fragment defined above. In the present context"replicable" means that the vector is able to replicate in a given typeof host cell into which it has been introduced. Immediately up-stream ofthe sequence encoding the polypeptide, the DNA may be provided with asequence encoding a signal peptide, the presence of which ensuressecretion of the polypeptide expressed by host cells harbouring thevector, as well as a cell harbouring the vector.

Also, an aspect of the invention is a method of producing a polypeptideas defined above comprising inserting a DNA fragment as defined aboveinto a vector which is able to replicate in a host cell, introducing theresulting recombinant vector into the host cell, culturing the host cellin an appropriate culture medium under appropriate conditions forexpressing the polypeptide, and recovering the polypeptide from the hostcell or culture medium.

The medium used to grow the cells may be any conventional mediumsuitable for the purpose. Furthermore, the polypeptide may be producedin a transgenic animal, e.g. a transgenic mammal producing thepolypeptide in the milk.

The DNA sequence used in the method may have been modified bysite-specific mutagensis, such as modified in that at least onenucleotide has been inserted, deleted, substituted or added to thesequence.

Furthermore, the present invention relates to a method of vaccinatingone or more selected persons of a population against tuberculosis andsubsequently subjecting the population to diagnostic tests fortuberculosis, comprising vaccinating the persons with a vaccine, whichcomprises as its effective component the above-discussed immunogenicagent, and subsequently subjecting the population to intradermalinjection of pharmaceutical compositions containing a polypeptide withwhich lymphoid cells previously primed with mycobacteria from thetuberculosis complex are capable of reacting in vitro and with whichlymphoid cells previously primed with the immunogenic agent are notcapable of reacting or a variant which is immunologically equivalent tothe polypeptide, whereby a positive skin response at the location ofinjection is indicative of the person having tuberculosis, and anegative skin response at the location of injection is indicative of theperson not having tuberculosis, and to the use of a vaccine, whichcomprises as its effective component the above-discussed immunogenicagent (e.g. BCG strain: Danish 1331), for vaccinating, againsttuberculosis, one or more persons of a population which subsequently isto be subjected to tuberculosis diagnosis using a diagnostic agentcomprising a polypeptide with which lymphoid cells previously primedwith mycobacteria from the tuberculosis complex are capable of reactingin vitro and with which lymphoid cells previously primed with theabove-discussed immunogenic agent are not capable of reacting or avariant which is immunologically equivalent to the polypeptide.

By this method there is no risk that the vaccination interferes with theskin testing for diagnosing tuberculosis because with the combination ofthe BCG strain used for vaccine purpose and the diagnostic skin test,positive Dth reactions with only occur in persons suffering fromtuberculosis, whereas no Dth reaction can be observed in personspreviously vaccinated and not suffering from tuberculosis.

DETAILED DESCRIPTION OF THE INVENTION

The importance of the polypeptide of the invention is its ability ofeliciting a delayed type hypersensitivity (Dth) reaction in personshaving active tuberculosis caused by virulent mycobacteria belonging tothe tuberculosis complex, but not eliciting a Dth reaction in personspreviously vaccinated with a vaccine comprising the above-discussedimmunogenic agent, e.g. BCG strain: Danish 1331. The Dth reaction is aninflammatory reaction occurring in the subcutaneous environmentexhibiting the cardinal features: erythema and induration due tocellular infiltration and edema. The diameter of this reaction ismeasured by visual inspection and use of a ruler.

The kit according to the invention will thus be useful in assessing aperson's immune status with respect to tuberculosis before vaccination,either to diagnose tuberculosis or to diagnose that the person has beenvaccinated with a vaccine different from the vaccine defined above. Thisis useful, e.g. at immigration camps where the immune status of theimmigrant's are tested before they are allowed immigration.

Furthermore, the kit may be useful for vaccinating individuals of apopulation and subsequently follow their immune status with respect totuberculosis infections, because the immunization caused by thevaccination does not give rise to a positive response to the skin test.Only persons having tuberculosis will have a positive response whensubjected to the skin test. Vaccinated persons may be subjected to theskin test periodically, such as every year or every second year, butother intervals may also be suitable depending on the population to betested.

In the present context the wording "immune status with respect totuberculosis" means whether the person in question has a positive ornegative immune response, when measured with the skin test of thepresent invention, which skin test is specific for tuberculosisinfection and therefore gives a specific picture of their immune status,i.e. whether they have tuberculosis or not.

The kit may comprise several skin tests, such as 3 or 5 skin tests,whereby the kit may be used for several years after the vaccination.

Furthermore, by using the above defined method of diagnosingtuberculosis it is thus possible to follow disease transmission rate byskin testing surveys in populations by subjecting the persons of thepopulation to a diagnostic skin test as defined above or as a diagnostictool in individual cases, and thereby diagnose the person(s) sufferingfrom active tuberculosis without having positive results from personspreviously vaccinated and not having active tuberculosis. Thus, thepolypeptide is one that is capable of reacting with lymphoid cells thatpreviously have been primed with mycobacteria belonging to thetuberculosis complex and one that is not capable of reacting withlymphoid cells that previously have been primed with mycobacteria fromthe above-discussed immunogenic agent, e.g. BCG strain: Danish 1331. Itis contemplated that this difference in reactivity between mycobacteriabelonging to the tuberculosis complex and those belonging to the BCGstrain: Danish 1331 is caused by the fact that virulent replicatingmycobacteria belonging to the tuberculosis complex actively secrete aprotein which is identical or immunologically equivalent to thepolypeptide, whereas replicating mycobacteria from the BCG strain:Danish 1331 do not secrete the protein or secrete the protein in such asmall amount that it cannot evoke a lymphoid immune response. However,mycobacteria from some of the other BCG strains used for vaccinepurposes may induce reactions similar to mycobacteria from the BCGstrain: Danish 1331 either because the mycobacteria do not secrete theprotein or if they secrete the protein in sufficient amounts to evoke alymphoid cell immune response, this immune response will not give riseto a persisting Dth reaction but fade out some time after thevaccination which in practice means that when vaccinated persons aretested no Dth reaction is elicited.

A method of measuring cellular immunity against the polypeptide, i.e.measuring whether the polypeptide reacts with lymphoid cells previouslyprimed may be carried out either in an in vitro system or an in vivosystem.

One in vitro system may be a lymphocyte proliferation assay. In thisassay peripheral blood mononuclear cells from persons vaccinated with avaccine comprising as its effective component the above-discussedimmunogenic agent and from persons having tuberculosis are co-culturedfor 4 to 5 days in the presence of the polypeptide as antigen. Immunelymphoid cells will proliferate in response to the antigenic stimulusand the proliferation is quantitated by the addition to the culture of3-H thymidine which will be incorporated in the DNA during cellreplication and measuring the amount of 3-H thymidine.

An in vivo system may be measurement of the Dth reaction occurring about24 to 48 hours after intracutaneous or intradermal injection ofmycobacterial antigen in a person or animal.

In the present context the term "immunologically equivalent variant,analogue or subsequence" means a variant, analogue or subsequence of thepolypeptide, which is capable of reacting with lymphoid cells primed asdescribed above and eliciting responses which are substantiallyidentical to the responses elicited by the polypeptide itself, oreliciting responses which are at least 45% identical to the responseselicited by the polypeptide itself.

When the kit and the method of diagnosing tuberculosis is used, the skinresponse should be measured a few days after the intradermal injectionhas been performed. The skin response mostly appears 1-4 days after theinjection, such as 2-3 days. If a skin response is observed and haswaned before 24 hours have passed after the injection, it is mostly dueto an irrelevant reaction which is not indicative of the person havingtuberculosis.

The skin response is measured as described above by visual inspectionand by the use of a ruler. A positive skin response is mostly between0.5 cm and 4.0 cm in diameter, more often between 1.0 cm and 3.0 cm indiameter.

An effective skin response is only obtained if a sufficient amount ofthe polypeptide remains at the location of injection; however, the sizeof some polypeptides may be so small that the polypeptide diffusesrapidly in the extracellular compartment at the site of injectionresulting in a less effective skin response. Consequently, as aspect ofthe present invention is a kit wherein the pharmaceutical compositioncomprises either a homopolymer or a heteropolymer of the polypeptide,whereby the polypeptide does not diffuse freely in the extracellularcompartment and is efficiently taken up by antigen-presenting cells atthe location.

A homopolymer of the polypeptide is to be understood in its usualmeaning, i.e. a polymer formed by two or more identical polypeptides,whereas a heteropolymer may be formed by at least two differentpolypeptides, or formed by a polypeptide and a heterologous carriermolecule.

The homopolymer may be formed by 2 or more copies of the polypeptide,such as 2-20 copies or 2-10 copies, more preferred 2-6 copies.

An example of the synthesis of a homopolymer may be the introduction ofone or more N-terminal cysteine residues in the polypeptide, therebyallowing the homopolymer to be formed as a result of intermoleculardisulphide bridges.

The synthesis of a heteropolymer may be carried out by coupling thepolypeptide to another mycobacterial polypeptide, such as themycobacterial protein MPT59 or part thereof (The MPT59 protein isdescribed in Nagai et al, Inf. and Imm. pp. 373-382, 1991).

By the synthesis of polymers of the polypeptide the specific activity orpotency will increase because the polypeptide will not diffuse freely inthe extracellular compartment, whereby a smaller dose of the polypeptideis necessary to elicit an observable Dth reaction.

Other kinds of modifications of the polypeptide may be relevant in orderto increase the activity of it. Such modifications may bepost-translational modifications such as acylation, i.e. addition of alipid moiety, and/or glycosylation.

In the kit according to the invention the pharmaceutical compositioncomprises 0.05 to 20 μg of the polypeptide, such as 0.5 to 2.0 μg of thepolypeptide, most preferred 0.75 to 1.5 μg of the polypeptide. When thepharmaceutical composition comprises polymers of the polypeptide thesame amounts are suitable.

In a preferred embodiment of the invention the amino acid sequence ofthe polypeptide comprises an amino acid sequence which is homologous tothe amino acid sequence shown in SEQ ID NO: 2, which is the sequence ofMPT64, including the sequence of the signal peptide, or homologous tothe amino acid sequence of an immunologically equivalent variant of thepolypeptide.

MPT64 is a protein which is secreted and released from metabolizingmycobacteria, in particular mycobacteria from the tuberculosis complex.

MPT 64 has an amino acid sequence of 205 amino acids with a calculatedmolecular weight of 22,433.

The polypeptide may also be a variant of the polypeptide with the aminoacid sequence shown in SEQ ID NO: 2, in that the amino acid sequence ofthe variant is homologous to an analogue or a subsequence of the aminoacid sequence shown in SEQ ID NO: 2.

The term "homologous" is used here to illustrate the degree of identitybetween the amino acid sequence of a given polypeptide and the aminoacid sequence shown in SEQ ID NO: 2. The amino acid sequence to becompared with the amino acid sequence shown in SEQ ID NO: 2 may bededuced from a DNA sequence, e.g. obtained by hybridization as definedabove, or may be obtained by conventional amino acid sequencing methods.The degree of homology is preferably determined on the amino acidsequence of a mature polypeptide, i.e. without taking any leadersequence into consideration. It is preferred that the degree of homologyis at least 80%, such as at least 90%, preferably at least 95% or even98% with the amino acid sequence shown in SEQ ID NO: 2.

Each of the polypeptides may be characterized by specific amino acid andnucleic acid sequences. It is to be understood, however, that suchsequences include analogues and variants produced by recombinant methodswherein such nucleic acid and polypeptide sequences have been modifiedby substitution, insertion, addition and/or deletion of one or morenucleotides in said nucleic acid sequences to cause the substitution,insertion, addition or deletion of one or more amino acid residues inthe recombinant polypeptide. When the term DNA is used in the following,it should be understood that for the number of purposes where DNA can besubstituted with RNA, the term DNA should be read to include RNAembodiments which will be apparent for the man skilled in the art.

In order to possess an ability of eliciting a Dth reaction a polypeptidemust be at least 12 amino acids long, preferably at least 15 aminoacids, such as 20 amino acids.

The polypeptide may have been encoded by a nucleotide sequencecomprising a nucleotide sequence homologous to the nucleotide sequenceshown in SEQ ID NO: 1, which is the nucleotide sequence encoding MPT64or a variant or analogue or subsequence of the nucleotide sequence, thevariant, analogue or subsequence encoding an immunologically effectiveequivalent to the polypeptide.

The variant or analogue refers to an nucleotide sequence wherein atleast one nucleotide has been substituted, deleted, inserted or addedeither leading to a modified amino acid sequence or to the same aminoacid sequence.

A nucleotide subsequence as used above refers to an effectivesubsequence which means that it encodes a polypeptide which isimmunologically functional with respect to the ability of eliciting aDth reaction. The subsequence may be the result of a truncation ateither end of the DNA sequence and/or of the removal of one or morenucleotides or nucleotide sequences within DNA sequence.

Interesting subsequences or deletion mutants are those shown in FIG. 10,and furthermore those constructed by use of the oligonucleotides shownin table 1 as primers in a PCR reaction as described in example 4.

The relevant functional parts of the polypeptide with respect to theability of the polypeptide to elicit a Dth reaction are the lymphoidcell epitopes. i.e. the parts of the amino acid sequence that arerecognized by lymphoid cells. These epitopes may either be linear orstructural.

The injection of the polypeptide may lead to an undesired sensitizationof the persons diagnosed for tuberculosis if the same person will besubjected to the skin test more than twice, or in extreme situationsmore than once.

Consequently, an object of the present invention is a kit wherein thepolypeptide has been modified in order to abolish or delete sensitizingepitopes, without abolishing the epitopes that are relevant with respectto the Dth reactions.

This may be carried out by several methods well-known to the personskilled in the art. One method may be to modify the polypeptide bydenaturing procedures, such as those selected from the group consistingof autoclaving or formaldehyde treatment.

Another method may be to modify the nucleotide sequence encoding thepolypeptide in such a way that the translated amino acid sequence lacksall or some of the sensitizing epitopes.

In the present context the wording "sensitizing epitopes" means epitopesthat cause sensitization of a person when the skin test has been usedfor diagnostic purposes. These epitopes may be either B-cell epitopes orT-cell epitopes.

Due to genetic variation persons may be divided into responders andnon-responders to a specific polypeptide based on their ability ofraising a lymphoid cell immune response to the polypeptide. Thus, forsome polypeptides a skin test wherein only one polypeptide is presentmay give rise to false negative responses, i.e. negative responses eventhough the person is suffering from tuberculosis because the lymphoidcell immune system of the person has not been able to raise an immuneresponse towards the polypeptide. Consequently, in a preferredembodiment of the present invention, the pharmaceutical compositioncomprises at least two different polypeptides either separated or aspolymers as described above, all the polypeptides being as definedabove.

One combination of polypeptides according to the invention is apharmaceutical composition wherein one polypeptide is MPT64 or animmunologically effective equivalent thereto, and another polypeptide isMPT59 or an immunologically effective equivalent thereto.

A pharmaceutical composition according to the invention is a compositionsuitable for intradermal injection.

The DNA fragment of the invention comprises a subsequence or a analogueof the nucleotide sequence shown in SEQ ID NO: 1, the subsequence oranalogue encoding a polypeptide which is immunologically equivalent tothe polypeptide encoded by the DNA sequence shown in SEQ ID NO: 1.

The subsequence and analogue are intended to be understood as definedabove.

Furthermore, a replicable expression vector comprising a DNA fragment asdescribed above is an aspect of the invention.

The vector may be any vector which may conveniently be subjected torecombinant DNA procedures, and the choice of vector will often dependon the host cell into which it is to be introduced. Thus, the vector maybe an autonomously replicating vectro, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication; examples of such a vector are a plasmid, phage,cosmid, mini-chromosome or virus. Alternatively, the vector may be onewhich, when introduced in a host cell, is integrated in the host cellgenome and replicated together with the chromosome(s) into which it hasbeen integrated.

The present invention further relates to a cell harbouring a replicableexpression vector as defined above. In principle, this cell may be ofany type of cell, i.e. a prokaryotic cell such as a bacterium, e.g. E.coli, a unicellular eukaryotic organism, a fungus or yeast, or a cellderived from a multicellular organism, e.g. an animal or a plant. It isespecially in cases where glycosylation is desired that a mammalian cellis used, although glycosylation of proteins is a rare event inprokaryotes.

In further aspect the present invention relates to the polypeptidecomprising an amino acid sequence which is different from but homologousto a subsequence, an analogue or a variant of the amino acid sequenceshown in SEQ ID NO: 2, the polypeptide being immunologically equivalentto the polypeptide of the amino acid sequence shown in SEQ ID NO: 2, thedegree of homology being as defined above.

Particularly interesting is a polypeptide which has been modified inorder to abolish or delete B- or T-cell epitopes which may besensitizing, without abolishing the epitopes that are relevant for theDth reaction.

In a yet further aspect the present invention relates to a method ofproducing a polypeptide as defined above, by inserting a DNA fragment asdefined above into a vector which is able to replicate in a host cell,introducing the resulting recombinant vector into the host cell,culturing the host cell in an appropriate culture medium underappropriate conditions for expressing the polypeptide, and recoveringthe polypeptide from the host cell or culture medium.

The medium used to grow the cells may be any conventional mediumsuitable for the purpose. A suitable vector may be any of the vectorsdescribed above, and an appropriate host cell may be any of the celltypes listed above. The methods employed to construct the vector andeffect introduction thereof into the host cell may be any methods knownfor such purposes within the field of recombinant DNA.

The DNA sequence to be modified may be of cDNA or genomic origin asdiscussed above, but may also be of synthetic origin. Furthermore, theDNA sequence may be of mixed cDNA and genomic, mixed cDNA and syntheticor genomic and synthetic origin as discussed above. The DNA sequence mayhave been modified, e.g. by site-directed mutagenesis, to result in thedesired DNA fragment encoding the desired polypeptide. The followingdiscussion focused around modifications of DNA encoding the polypeptideshould be understood to encompass also such possibilities, as well asthe possibility of building up the DNA by ligation of two or more DNAfragments to obtain the desired DNA fragment, and combinations of theabove-mentioned principles.

The DNA sequence may be modified using any suitable technique whichresults in the production of a DNA fragment encoding a polypeptide ofthe invention.

The modification of the DNA sequence encoding the amino acid sequence ofthe polypeptide of the invention should be one which does not impair theimmunological function of the resulting polypeptide.

Also, the polypeptide of the invention may be produced by the well-knownmethods of solid or liquid phase peptide synthesis utilizing thesuccessive coupling of the individual amino acids of the polypeptidesequence or coupling of individual amino acids forming fragments of thepolypeptide sequence so as to result in the desired polypeptide.

The design of skin test for diagnosing tuberculosis, which is closelyrelated to a vaccine for vaccinating persons against tuberculosis, sothat a minimum of false positive indication of tuberculosis are detectedis an aim of the present invention. This has been fulfilled by thedescribed kit comprising a skin test and a vaccine comprising as theeffective component the above-discussed immunogenic agent, because thepolypeptide of the method does not give rise to a Dth reaction in aperson, if the person has been immunized with a vaccine comprising theimmunogenic agent.

Consequently, an effective population survey may be conducted if theindividuals of the population have been vaccinated with the describedvaccine and the subsequent diagnostic tests are carried out by themethods as described.

Thus, an object of the invention is a method of vaccinating one or moreselected persons of a population against tuberculosis and subsequentlysubjecting the population to diagnostic tests for tuberculosis,comprising vaccinating the persons with a vaccine, which comprises asits effective component the above-discussed immunogenic agent, andsubsequently subjecting the population to intradermal injection ofpharmaceutical compositions containing a polypeptide with which lymphoidcells previously primed with mycobacteria belonging to the tuberculosiscomplex are capable of reacting in vitro and with which lymphoid cellspreviously primed with the immunogenic agent are not capable of reactingor a variant which is immunologically equivalent to the polypeptide,whereby a positive skin response at the location of injection isindicative of the person having tuberculosis, and a negative skinresponse at the location of injection is indicative of the person nothaving tuberculosis.

Another object of the invention is the use of a vaccine which comprisesas its effective component the above-discussed immunogenic agent forvaccinating, against tuberculosis, one or more persons of a populationwhich subsequently are to be subjected to tuberculosis diagnosis using adiagnostic agent comprising a polypeptide with which lymphoid cellspreviously primed with mycobacteria belonging to the tuberculosiscomplex are capable of reacting in vitro and with which lymphoid cellspreviously primed with the immunogenic agent are not capable of reactingor a variant which is immunologically equivalent to the polypeptide.

LEGENDS TO FIGURES

FIG. 1: The sequence of the gene encoding MPT64 and the correspondingdeduced amino acid sequence of MPT64. The first arrow indicates thestart of the polypeptide including the signal sequence, the second arrowindicates the sequence of the mature protein when the signal sequencehas been cleaved of. Furthermore, the start position of theoligonucleotide sequences shown in table 1 is shown, as well as therestriction sites discussed in example 4.

FIG. 2: The distribution of skin reactions to mycobacterial antigens.

Groups of 8 Ssc:AL guinea pigs were infected i.v. with M. tuberculosisH37Rv or with BCG strain: Danish 1331 given i.d. or i.v. Three weekslater guinea pigs were skin tested with 1 μg of the indicated purifiedantigens and with 10 T.U. of tuberculin.

FIG. 3: The distribution of skin reactions to mycobacterial antigens.

Groups of guinea pigs of 8 Ssc:AL and of strains NSD and IMM/R wereinfected i.v. with M. tuberculosis R1609 and skin tested 3 weeks laterwith 1 μg of the indicated purified antigens and 10 T.U. of tuberculin.

FIG. 4: The distribution of skin reactions to mycobacterial antigens.

Groups of 10 Ssc:AL guinea pigs were infected i.v. with M. tuberculosisH37Rv, BCG strain: Danish 1331 or immunized with killed M. tuberculosisin oil and skin tested 3 weeks later with 1 μg of the indicated purifiedantigens and 10 T.U. of tuberculin.

FIG. 5: The peripheral blood lymphocyte stimulation results to 1 μg ofthe mycobacterial preparations.

Groups of Ssc:AL guinea pigs were sensitized by infection with M.tuberculosis H37Rv, BCG, i.d., BCG i.v. or immunized with killed M.tuberculosis in oil or with the oil (M52) alone. HBT12 designates the 38kDa antigen. Results are shown as means within immunization groups forstimulation indices. Indices above 3 are considered significant.

FIG. 6: The distribution of skin reactions to BCG antigens. Groups of 10Ssc:AL guinea pigs were vaccinated i.d. with BCG strain: Danish 1331 orBCG Tokyo and skin tested 3 weeks later with 1 μg of the indicatedpurified antigens and 10 T.U. of tuberculin.

FIG. 7: The distribution of skin reactions to mycobacterial antigens.

Groups of 8 Ssc:AL guinea pigs were infected with M. tuberculosis R1609or M. bovis MNC27 and skin tested 3 weeks later with 1 μg of theindicated purified antigens and with 10 T.U. of tuberculin.

FIG. 8: The distribution of skin reactions in three groups of Ssc:ALguinea pigs.

Three groups of 10 Ssc:AL guinea pigs were infected with M. tuberculosisR1609 and/or BCG strain: Danish 1331. Group 1: BCG, skin tested after 10weeks. Group 2: No BCG, skin tested 3 weeks after M. tuberculosisinfection. Group 3: BCG, M. tuberculosis infection after 7 weeks, skintested after 10 weeks. All groups were skin tested simultaneously with 1μg of the purified antigens indicated and with 10 T.U. of tuberculin.

FIG. 9: The distribution and (solid arrows) of reactions in four groupsof Ssc:AL guinea pigs.

Four groups of 7-8 Ssc:AL guinea pigs were infected with M. tuberculosisH37Rv. Three weeks later one group was skin tested and the otherstreated with antibiotics. The remaining groups were skin tested after 6,10 and 14 weeks, one group at each time point. Skin tests were done with1 μg of MPT64 and with 10 T.U. of tuberculin.

FIG. 10: Deletion mutants constructed as described in example 4.

FIG. 11: Southern blot showing reaction with nick-translated recombinantMPT64 DNA.

Lane 1: M. bovis BCG Tokyo, lane 2: M. bovis BCG Moreau, lane 3: M.bovis BCG Russian, lane 4: M. bovis BCG Glaxo, lane 5: M. bovis BCGPasteur, lane 6: M. bovis BCG Canadian, lane 7: M. bovis BCG Tice, lane8: M. bovis BCG Copenhagen, lane 9: M. tuberculosis H37Rv, lane 10: M.tuberculosis H37Ra, lane 11: M. tuberculosis Erdman, lane 12: M. leprae.

FIG. 12: The skin test inducing capacity of recombinant MPT64 (rMPT64)after removal of the fusion partner (the maltose binding protein).

The skin test inducing capacity of rMPT64 (0.1 μg) was compared tonative MPT64 and PPD RT23 (2 TU) by assessing the reactions in groups(n=8) of outbred guinea pigs (Ssc:Al) immunized with either M. bovis BCGTokyo (black bars) or M. bovis BCG Copenhagen (open bars). Skin testingwas performed 4 weeks after immunization.

FIG. 13: Physical map of recombinant plasmids expressing various regionsof mpt64 and skin reactions in guinea pigs to fusion proteins expressedby the plasmids.

Left part of the figure: The open bars are vector DNA. the closed bar ismpt64. The transcription of the gene is from left to right.

Right part of the figure: The reactivity against semi-purifiedrecombinant fusion proteins established by skin testing of guinea pigsimmunized with BCG Danish 1331 (left column) and BCG Tokyo (rightcolumn). A `+` indicates a skin reaction larger than 8 mm, a `-`indicates a skin reaction of less than 4 mm, and `ND` indicates that thereaction not has been determined.

FIG. 14: Skin test reactions obtained in outbred guinea pigs by purifiedrecombinant MPT64 fused to the maltose binding protein and mutatedversions thereof lacking either parts of the N-terminus or theC-terminus of the protein as shown on the physical map in FIG. 13.

The reactions obtained by the maltose binding protein alone (producedand purified in the same way from the construct designated pTO13) didnot exceed 4 mm. The concentration of contaminating lipopolysaccharidewas determined by a conventional limulus assay and was found to compriseless than 0.05 ng/μg protein. It was concluded from this experiment thatthe biological activity of MPT64 is contained within the carboxyterminalone third of the protein (downstream from the endpoint of TO21).Restriction sites: C: ClaI; E. EcoRI; S: StuI; Sa: SalI: Sm: SmaI.

FIG. 15: Amino acid sequences of synthetic peptides used in skin testsin example 7.

The peptides are composed of 25 amino acids derived from the deducedamino acid sequence in FIG. 1.

FIG. 16: Overlap of the synthetic peptides from FIG. 15 and skin testresults after injection with the synthetic peptides.

The histogram shows the delayed type hypersensitivity reactions elicitedin groups of outbred guinea pigs (n=8) after intradermal injections of10 μg of synthetic peptides derived from the carboxyterminal one thirdof the MPT64. The guinea pigs were either immunized with M. bovis BCGTokyo or BCG Danish 1331. The skin tests were performed three to fourweeks after the immunization with the synthetic peptides of FIG. 15 andoverlapping as indicated by solid lines marked: A1, A2, A3, A4, B1, B2,B3, B4, C1, C2, C3, C4, D1, D2, and D3. The reactions were read after 24hours by two independent technicians.

FIG. 17: Diameters in skin reaction following injection of 1 μg MPT64 inguinea pigs previously infected with aerosols of M. tuberculosis Erdman.

FIG. 18: Diameters in skin reaction following injection of 10 TUtuberculin in guinea pigs previously infected with aerosols of M.tuberculosis Erdman.

Each of the symbols in FIGS. 17 and 18, respectively, represents oneindividual; e.g., in FIG. 17 a solid diamond represents the skinreaction diameter in the same guinea pig at different time points afterinfection.

EXAMPLES

The following examples are intended to illustrate but not to limit thepresent invention.

The following methods are used generally in the following examples andare therefore discussed generally:

Hybridization of DNA

DNA, e.g. present on nitrocellulose filters, are wetted in 2×SSC (1×SSC:0.15 M NaCl, 0.0015 M Na₃ -citrate, pH 7.0) and placed in a heat-sealedplastic bag with pre-warmed (65° C.) prehybridization solution.Prehybridization takes place for 2 h at 65° C., the bag being gentlyshaken. The solution is exchanged with pre-warmed (65° C.) hybridizationsolution, a radioactive probe is added and hybridization is carried outat 65° C. for 18 h. The bag is gently shaken to ensure constant movementof the liquid over the nitrocellulose filters. After hybridization, awashing procedure is carried out.

The radioactive probe is prepared by use of known methods, e.g. asdescribed by Sambrook et al., on the basis of the DNA sequence shown inSEQ ID NO: 1 or a part thereof, especially a coding part or an effectivesubsequence of the DNA sequence as defined above.

The prehybridization and hybridization solutions used are:10×Denhardt's, 4×SSC, 0.1% SDS, 10 μg/ml polyA, 50 μg/ml of denaturedDNA to be analyzed and the denatured (heat) radioactive probe. Thefilters are washed in pre-warmed (67° C.) solutions: 10×Denhardt, 2×SSC,0.1% SDS for 2×15 min. and 1×SSC, 0.1% SDS for 4×15 min. The filters areair-dried and covered with Vita-Wrap, and X-ray film is exposed to thefilters for 3 h to 3 weeks with and without intensifying screens.

EXAMPLE 1

Skin testing

MATERIALS AND METHODS

Guinea pigs

Outbred guinea pigs from strains Ssc:AL, Bfa/ZH/Kissleg and Hsd/Poc:DHwere used. Additionally, two inbred strains NSD and IMM/R were used.

Bacterial strains

The following strains were used: M. tuberculosis H37Rv, M. tuberculosisR1609 (isolated from a Danish patient with lung tuberculosis), M.tuberculosis Erdman, M. bovis MNC27, M. bovis BCG strain: Danish 1331,M. bovis BCG Tokyo.

Infection and immunization of guinea pigs

When infected with strains of M. tuberculosis or virulent M. bovisguinea pigs were given 2.5×10³ cfu in a volume of 0.1 ml in an ear vein.Infection by the same route (i.v.) with BCG was done with 2.5×10⁴ cfu.Vaccinations with BCG were done with four intradermal (i.d.) injectionson the abdomen of 0.1 ml of reconstituted BCG vaccine. BCG strain:Danish 1331 contained approximately 4×10⁶ and BCG Tokyo 24×10⁶ cfu perml of the reconstituted preparations. Immunizations with killed bacteriawere given 4×0.1 ml i.d. on the abdomen of a suspension ofglutaraldehyde killed bacteria at 0.4 mg (semidry weight) per ml ofparaffine oil (Marcol 52 (M52)).

Antiobiotic therapy

When indicated in the text, guinea pigs were given isoniazide (INH)(Merck) at 100 mg/l and rifabutin (R) (Farmitalia Carlo Erba) at 100mg/l in the drinking water.

Bacterial enumeration

The number of viable bacteria in the spleens of infected mice wasdetermined by plating double serial ten-fold dilutions of organhomogenates on Lowenstein-Jensen medium. Colonies were counted after 3to 4 weeks of incubation. The results presented are geometric meanswithin groups.

Antigens

Tuberculin PPD RT23 (Statens Seruminstitut) was used as a positivecontrol in skin test experiments in sensitized guinea pigs.

MPT59 and MPT64 were prepared by S. Nagai as described by Nagai et al.(1991). M. tuberculosis H37Rv were cultured for 5 weeks on Sauton mediumand the culture supernatant harvested by centrifugation. The supernatantwas concentrated by precipitation with 80% ammonium sulphate. Theconcentrate was applied to a DEAE-Sepharose CL-6B (Pharmacia, Uppsala)column with 30 mM Tris hydrochloride buffer (pH 8.7) with 3%methylcellosolve. The proteins were eluted with a sodium chloridegradient. MPT59 and MPT64 is primarily contained in the fraction elutedwith 110-140 mM sodium chloride. This fraction was applied to aDEAE-Sepharose CL-6B column in 30 mL Tris hydrochloride (pH 7.5) with 3M urea, and was eluted with 50-90 mM NaCl. The eluate was divided andapplied to two Sephacry S-200 HR columns in 10 mM Tris hydrochloride (pH7.5) with 10% ethylene glycol and 300 mM NaCl. The relevant fractionsfrom the S-200 runs containing MPT64 or MPT59, respectively, wereapplied to Phenyl-Sepharose CL-48 columns, the MPT64 fraction in 10 mMTris hydrochloride (pH 7.5) and the MPT59 fraction in the same bufferwith 50 mM ammonium sulphate (AS).

The MPT64 column was eluted with an AS gradient. The fraction 200 mM -25 mM AS was applied to DEAE-Sepharose CL-6B in 30 mM Tris-HCl (pH 8.7).MPT64 was eluted with 60-90 mM NaCl.

The MPT59 column was washed with 10 mM Tris-HCl (pH 7.5) and eluted with10 mM Tris-HCl (pH 8.7). The eluate containing MPT59 was applied toDEAE-Sepharose CL-6B in 30 mM Tris-HCl (pH 8.7). MPT59 was eluted with80-110 mM NaCl.

The 38 kDa antigen was purified by affinity chromatography as describedpreviously (Worsaae et al., 1987). M. tuberculosis H37Rv was grown onSauton medium and the culture filtrate (CF) isolated by filtration. CFwas precipitated twice with 80% ammonium sulphate, redissolved anddialysed against phosphate buffered saline (pH 7.4). The affinity columnwas prepared by coupling the HBT12 monoclonal antibody to CNBr activatedSepharose 4B (Pharmacia). CF was passed through the column at 1-5 mg/mlof phosphate buffered saline (pH 7.4) with 0.5 M NaCl and 0.05% Tween20. After washing the antigen was eluted with 0.1 M glycinehydrochloride (pH 2.8) and dialysed against phosphate buffered saline(pH 7.4).

Skin tests

Guinea pigs were given i.d. injections on the shaven back of 0.1 mlphysiological phosphate buffered saline (pH 7.4) containing the desiredquantity of antigen. Guinea pigs were depilated on the back 24 h laterand reactions were read by two independent readers, each measuring twotransverse diameters of the erythemas. Reaction diameters are given asmeans corresponding to a single diameter.

Lymphocyte stimulation tests

Peripheral blood lymphocytes were isolated from blood drawn by cardiacpuncture using EDTA as anticoagulant. Erythrocytes were removed byficoll density gradient (d=1.09) centrifugation. Lymphocytes were washedtwice, counted and the cell concentration adjusted to 2×10⁶ cells/ml inRPMI 1640 with supplements including 5% FCS. Spleen lymphocytes wereisolated by pressing spleens through a wire mesh. Erythrocytes werelysed by treatment with 0.84% NH₄ Cl. The lymphocytes were washed twiceand the cell concentration adjusted to 2×10⁶ cells/ml of RPMI withsupplements.

0.1 ml of cells were cultured with 0.1 ml of antigen or mitogen intriplicate for 6 days, the last 22 h in the presence of 1 μCi ³H-thymidine. Cultures were harvested and incorporated ³ H-thymidinecounted in a scintillation counter. Results were expressed asstimulation indices using geometric means of triplicate cultures, thestimulation index being defined as the ratio between stimulated andnon-stimulated cultures.

RESULTS

MPT64 skin tests distinguish infections with M. tuberculosis and BCG

Two separate experiments were carried out to compare skin reactivity toselected mycobacterial antigens in groups of Ssc:AL guinea pigs (GP's)infected i.v. or vaccinated i.d. with BCG strain: Danish 1331 toreactivity in animals infected with M. tuberculosis (H37Rv). Skin testswere applied 3 weeks after infection/vaccination. The results, which aresummarized in FIG. 2, show that BCG as well as M. tuberculosis inducesimilar reactions to 10 T.U. of tuberculosis and to the 38 kDa antigen.In contrast, M. tuberculosis infection induces large reactions to MPT64in the majority (approximately 70%) of GP's, but not in any BCG primedGP. The minority of M. tuberculosis infected GP's are, most likely forgenetic reasons non-responders to MPT64. The majority of M. tuberculosisinfected GP's are non-responders to MPT59.

Genetic restriction of MPT64 responses

The presence of non-responders to a diagnostic reagent in a humanpopulation is of course an issue of great concern.

To pursue this question, two additional outbred strains of GP's wereinfected with M. tuberculosis or BCG i.v. and skin tested as describedabove. The results (not shown) were similar to those obtained withSsc:AL GP's, in particular a similar low frequency of non-responders wasseen.

In another experiment, two inbred strains of GP's, NSD and IMM/R wereinfected with M. tuberculosis and skin tested. The results show thatboth strains are uniform responders to MPT64, but non-responders toMPT59 (FIG. 3).

Clinical experiments will clarify whether and to what extent humans arenon-responders to these antigens.

Comparisons of MPT64 reactivity (in vivo and in vitro) in guinea pigssensitized with living and killed M. tuberculosis

Because MPT64 and MPT59 are secreted proteins, it is relevant to comparereactivity in GP's sensitized with living and killed bacteria. Groups ofGP's were infected with M. tuberculosis, BCG or immunized with killed M.tuberculosis in oil and skin tested 3 weeks later. The results show thatsimilar tuberculin reactions were induced by the differentsensitizations (FIG. 4). Positive skin reactions to MPT64 and MPT59were, however, absent in GP's immunized with killed M. tuberculosis, andas seen previously in BCG-vaccinated GP's.

In a similar experiment groups of Ssc:AL GP's were sensitized byinfection with M. tuberculosis, BCG i.d., BCG i.v., or immunized withkilled M. tuberculosis in oil or (as a control) oil alone (M52). 3 weekslater peripheral blood and spleen lymphocytes were isolated and used forlymphocyte stimulation experiments. The results obtained were similarfor both cell types. The results from peripheral blood cells shown inFIG. 5 demonstrate, with the exception of the control group, uniformlystrong responses to tuberculin PPD and uniformly moderate responses tothe 38 kDa antigen. In contrast, only lymphocytes from M. tuberculosisinfected GP's reacted to MPT64, thus confirming the skin test results.

The results suggest that growth of M. tuberculosis is a prerequisite fordevelopment of MPT64 reactivity.

Skin reaction to MPT64 in M. bovis and M. bovis BCG Tokyo infectedguinea pigs

To extend the observations on bacterial species and strain specificityof MPT64, reactivity was compared in groups of GP's vaccinated with BCGstrain: Danish 1331 and BCG Tokyo (FIG. 6) and in groups infected withM. tuberculosis or a virulent strain of M. bovis (FIG. 7).

The results show that vaccination with BCG Tokyo induces skin reactionsto MPT64 and MPD70, but not MPT59 (FIG. 6). Vaccination with BCG strain:Danish 1331 did not induce reactions to these antigens. The differencein MPB70 reactivity between these BCG strains, which belong to different"families", has been described previously.

Comparison of reactivity to MPT64 and MPT59 in M. bovis and M.tuberculosis infected GP's show similar distribution of skin reactionsin the groups. This suggests that MPT64 may be a useful diagnosticreagent in bovine tuberculosis.

MPT64 skin reactions in BCG vaccinated guinea pigs infected with M.tuberculosis. It is an important consideration in a diagnostic situationwhether a BCG vaccinated individual subsequently infected with M.tuberculosis develops reactivity to MPT64 whether or not diseasedevelops. Accordingly, three groups of GP's were given 2.5×10³ cfu BCGstrain: Danish 1331 and/or 2.5×10³ cfu M. tuberculosis as shown below:

    ______________________________________                                        BCG                                                                                            Skin tests                                                                    ↓ ↓ ↓                                   Group 1 -------------------------------------------------- →                        M. tub.                                                                             Skin tests                                                              ↓                                                                            ↓ ↓                                          Group 2 -------------------------------------------------- →           BCG            M. tub. Skin tests                                             ↓       ↓                                                                              ↓                                               Group 3 -------------------------------------------------- →           0              7       10                                                     ↓       ↓                                                                              ↓                                               Weeks  -------------------------------------------------- →            ______________________________________                                    

All guinea pigs were skin tested and spleens taken for counting of M.tuberculosis after 10 weeks. The results shown in FIG. 8 demonstratethat all groups of GP's develop similar tuberculin reactivity and thatMPT64 gives reactions only in group 2. It thus appears that preinfectionwith BCG before M. tuberculosis infection prevents sensitization toMPT64.

The mean results from counting of M. tuberculosis were:

Group 1: 0 cfu/spleen

Group 2: 3×10³ cfu/spleen

Group 3: 7.7×10⁵ cfu/spleen

Thus, the preinfection with BCG has resulted in a 200-fold reduction ofbacterial growth in the spleen. These results indicate that substantialgrowth of M. tuberculosis is a prerequisite for development of MPT64reactivity in the GP, and make it likely that disease is likewise aprerequisite in a patient.

Persistency of MPT64 reactivity

Persistency of reactivity is an important parameter in the evaluation ofa diagnostic reagent. To address this problem, 4 groups of GP's wereinfected with M. tuberculosis and 3 weeks later given isoniazide andrifabutin in the drinking water to prevent further growth of thebacteria. One group was skin tested after 3 weeks, one after 6, oneafter 10 and the last 14 weeks following constant throughout theobservation period, whereas the reactivity to MPT64 drops rapidly to alow plateau level (FIG. 9).

These results suggest that MPT64 may have its prime use in the diagnosisof an acute infection rather than as an epidemiological tool.

Conclusion and comments

The present series of experiments suggests that MPT64 may be a suitableskin test reagent for diagnosis of tuberculosis, a reagent with aspecificity not present in tuberculin preparations.

Another important issue for the general usefulness of the preparation ishow long BCG Tokyo vaccinated persons retain their vaccine-induced MPT64reactivity. If this reactivity has waned at a time they contract atuberculosis infection, MPT64 will be a useful diagnostic reagent insuch patients.

EXAMPLE 2

Cloning and expression of MPT64

DNA technology

Standard procedures were used for the preparation and handling of DNA(Maniatis, T., E. F. Fritsch, and J. Sambrook. 1989. Molecular Cloning.A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.).

Synthesis and design of probes

Oligonucleotide primers were synthesized automatically with a DNAsynthesizer (Applied Biosystems (ABI-391, PCR-mode)) and purified byethanol precipitation. Four oligonucleotides were synthesized on thebasis of the nucleotide sequence from MPB64 (Yamaguchi, R., K. Matsuo,A. Yamazaki, C. Abe, S. Nagai, K. Terasaka, and T. Yamada. 1989. Cloningand Characterization of the Gene for Imunogenic Protein MPB64 ofMycobacterium bovis BCG. Infect. Immun. 57:283-288), and fiveoligonucleotides were synthesized on the basis of the deduced nucleotidesequence from MPT64 in this study (Table 1). The oligonucleotides wereengineered to include an EcoRI restriction enzyme site at the 5' end andat the 3' end by which a later subcloning was possible.

                                      TABLE 1                                     __________________________________________________________________________    Sequence of the MPT64 oligonucleotides                                        Oligonucleotide                                                                         Sequences (5'-3')     Position                                      __________________________________________________________________________    Sense                                                                         MPT64-1   GAA TTC GCG CCC AAG ACC TAC TGC                                                                     207 to 225                                    MPT64-4   GAT GCG AAT TCG AAA ATT ACA TCG CCC                                                                 337 to 352                                    MPT64-5   GAT GCG AAT TCA AGG TCT ACC AGA ACG                                                                 479 to 496                                    MPT64-6   GAT GCG AAT TCC AGG CCT ATC GCA AGC                                                                 543 to 559                                    MPT64-7   GAT GCG AAT TCA GCA AGC AGA CCG GAC                                                                 637 to 652                                    MPT64-8   GAT GCG AAT TCG ACC CGG TGA ATT ATC                                                                 685 to 700                                    MPT64-9   CTC GAA TTC TGC TAG CTT GAG                                                                          1 to 14                                      Anti-sense                                                                    MPT64-2   GAA TTC TAG GCC AGC ATC GAG TCG                                                                     826 to 807                                    MPT64-3   GAA TTC CGG CGT TCT GGT AGA CC                                                                      500 to 483                                    __________________________________________________________________________     Oligonucleotides: MPT641, MPT642, MPT643, and MPT649 were constructed fro     the MPB64 sequence (Yamaguchi et al. 1989). The rest of the                   oligonucleotides were constructed after the nucleotide sequence obtained      from MPT64 reported in this work.  # The Oligonucleotides are listed in       SEQ ID NO: 3 to SEQ ID NO: 11.                                                Nucleotides underlined are not contained in the nucleotide sequence of        MPB64 or MPT64.                                                          

DNA cloning

MPT64 was cloned from M. tuberculosis H37Rv chromosomal DNA extractedand purified as described by Andersen et al. (Andersen Å. B., P.Andersen, L. Ljungquist 1992. Structure and Function of a 40,000Molecular Weight Protein Antigen of Mycobacterium tuberculosis. Infect.Immun. 60, 2317-2323) by the use of the PCR technology as described byInnis et al. (Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J.White. 1990. PCR Protocols. A Guide to Methods and Applications, pp.253-258. Academic Press, Inc., San Diego, Calif.).

In brief, the standard amplifications were carried out in a TermalReactor, Hybaid, Teddington, UK, by incubation of 100 ng of chromosomalM. tuberculosis H37Rv brought to a final volume of 37 μl with Milli Qwater at 70° C. for 5 minutes and then cooled on wet ice for 10 minutes.13 μl of PCR master mix was added. The PCR master mix contained: 192 mMKCl, 38.5 mM Tris/HCl, pH 8.3, 5.8 mM MgCl₂, 0.77 mM in each dNTP and3.8 μM in each oligonucleotide primer. The reaction mixture wereoverlayed with 100 μl mineral oil. Denaturation of the DNA was carriedout at 94° C. for 5 minutes. The reaction mix were brought to theannealing temperature, 60° C., 1.5 units AmpliTaq DNA polymerase (PerkinElmer Cetus, Norwalk, Conn.) was added to the master mix. Theamplifications were performed for 30 cycles, 72° C. for 3 minutes, 94°C. for 1 minute 20 seconds, 60° C. for 2 minutes. At the end of thecycles the primer extension step was carried out for 7 minutes.

10 μl of the PCR product was fractionated on 1.5% w/v agarose gelelectrophoresis and visualized with ethidium bromide. Negative controlscontaining all PCR reagents except DNA were run in parallel with thesamples.

The PCR product was cloned in the pCR1000 vector as described for the TACloning System (InVitrogen, San Diego, Calif.) and transformed E. coliINVaF' (EndA1, recA1, hsdR17 (r-k, m+k), supE44, λ-, thi-1, gyrA, relA1,ρ80 lacZΔM15Δ (lacZYA-argF9, deoR+, F') (InVitrogen, San Diego, Calif.).

Four oligonucleotides (Table 1) were constructed from the sequence ofMPB64 (Yamaguchi et al. 1989) in expectation of some sequence homologybetween MPB64 and MPT64. Hereby were i) the structural gene of MPT64 andii) the promoter region, the signal sequence and the N-terminal of MPT64comprised. The PCR reactions gave two specific bands at i) 628 bp, andii) 508 bp.

DNA Sequencing

The cloned 628 bp M. tuberculosis H37Rv PCR fragment, pTO1, containingthe structural gene of MPT64, and the cloned 508 bp PCR fragmentcontaining the promoter region and the signal peptide sequence, pTO3,was determined by the dideoxy chain termination method using a SequenaseDNA sequencing kit version 1.0 (U.S. Biochemical Corp., Cleveland, Ohio)according to the instructions provided. Both strands of the DNA weresequenced.

The nucleotide sequences of pTO01 and pTO03 of a gene coding MPT64 areshown in SEQ ID NO: 1 (and in FIG. 1).

Next, the nucleotide sequences were compared with the known amino acidsequence around N-terminus of MPT64 protein to find the GCG codon atposition 208-210 corresponding to the N-terminal amino acid, Ala, ofMPT64 (Andersen et al. 1991), and a TAG codon at position 823-825 is atermination codon. Therefore a structural gene coding for MPT64 proteinderived from M. tuberculosis H37Rv was found to correspond to position208-822 of the nucleotide sequence in SEQ ID NO: 1.

The nucleotide sequence for MPT64 contains only few nucleotidedifferences compared to MPB64. One nucleotide change is found in thestructural gen of MPT64, at position 453 where a G is converted to an A.From the deduced amino acid this change occurs at a third position ofthe amino acid which does not lead to changes of the amino acidsequence. In the signal sequence at position 198 a G is converted to aC, also without any changes in the deduced amino acid composition. Inthe non-structural region of the promoter, the Shine-Dalgarno, etc. twodifferences occur, one addition at position 47 of a C, and one deletionof a G at position 100.

Thus, it is concluded that the structural gene for MPT64 consists of 615bp and that the deduced amino acid sequence contains 205 amino acidswith a calculated molecular weight of 22,433.

EXAMPLE 3

Subcloning of the MPT64 gene

An EcoRI site was engineered immediately 5' of the first codon of thegene so that only the coding region of the gene encoding the M.tuberculosis H37Rv 24 kD protein would be expressed, and an EcoRI sitewas incorporated right after the stop codon at the 3' end.

DNA of the recombinant plasmid pTO1 was cleaved at the EcoRI sites. The628 bp fragment was purified from an agarose gel and subcloned into theEcoRI site of the pMAL-p expression vector (New England Ciolabs,Beverly, Mass.). Vector containing the gene fusion was transformed inthe E. coli XL1-Blue (Bullock, W. O., J. M. Fernandez, and J. M. Short.1987. XL1-Blue: A high efficiency plasmid transforming recA Escherichiacoli strain with beta-galactosidase selection. BioTechniques 5: 376-379)for expression by the standard procedures for DNA manipulation.

The endpoints of the gen fusion were determined by the dideoxy chaintermination method as described under the section DNA sequencing. Bothstrands of the DNA were sequenced.

EXAMPLE 4

Construction of MPT64 deletion mutants

DNA of the recombinant plasmid pTO1 was cleaved in the MPT64 gene at theClaI, the StuI or the SmaI site (FIG. 11). The DNA was treated with theKlenow fragment of DNA Polymerase I (Gibco BRL, Life Technology A/S,Roskilde, Denmark) to make the ends blunt. Subsequently the DNA wasdigested with EcoRI, and the 327 bp EcoRI--StuI, the 459 bp EcoRI--ClaI,and the 542 bp EcoRI--SmaI fragments were purified from a 2% w/v agarosegel.

The pMAL-p vector was cleaved at the unique SalI site, and the DNA wastreated with the Klenow fragment of DNA Polymerase I to make the endblunt. The DNA was afterwoards digested at the unique EcoRI site, andthe large EcoRI--SalI fragment was purified from a 0.8% w/v agarose gel.

Additional one C-terminal deletion mutant was engineered by PCR usingthe primers MPT64-1 and MPT64-3 (Table 1). The 299 by EcoRI digestedfragment was subcloned in pMAL-p.

The different construction possibilities were ligated. The ligated DNAwas transformed into E. coli XL1- Blue and plated on Luria-Bertani agarwith 50 μg/ml ampicillin, 12.5 μg/ml tetracycline, and 80 μg/ml5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal). White colonies werepicked randomly, and plasmid DNA was cleaved with BamHI and HindIII, andanalysed by agarose gel electrophoresis to control determine the size ofthe mycobacterial insert.

Deletion mutants containing DNA of the right sites were sequenced by thedideoxy chain termination method as described under the section DNAsequencing to confirm the inframe fusion to malE in pMAL-p. Both strandsof the DNA were sequenced in all the constructions.

To create deletion mutants from the N-terminal of the gene also, fiveoligonucleotides, MPT64-4, MPT64-5, MPT64-6, MPT64-7, and MPT64-8 (Table1), containing an EcoRI site were engineered to create an inframe fusionwith the malE of the pMAL-p vector by PCR as described in the DNAcloning section. The EcoRI digested PCR fragments were subcloned in theEcoRI site of the pMAL-p expression vector. A vector containing the genefusion was transformed into the E. coli XL1-Blue for expression bystandard procedures for DNA manipulation. To confirm that the deletionsof all five constructions were in frame with the malE gene in pMAL-p,both strands were sequenced by the dideoxy chain termination method asdescribed under the section DNA sequencing.

A physical map of a number of these deletion mutants can be seen in FIG.13.

EXAMPLE 5

Preparation and purification of recombinant MPT64 and truncated versionof MPT64

Recombinant antigens were prepared in accordance with instructionsprovided by New England Biolabs. Briefly, XL1-Blue cells containing thefusion plasmid of interest were grown in Luria-Bertani media with 50μg/ml ampicillin and 12.5 μg/ml tetracycline to A₆₀₀ nm app. 0.5, andthe production of the fusion protein was induced with 0.3 mMisopropylthiogalactoside (IPTG) at 37° C. for 2 hours. The pelletedXL1-Blue cells were frozen at -20° C. overnight in the column buffer (20mM TRIS/HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA, and 1 mM DTT) and thawed at4° C. followed by incubation with 1 mg/ml lysozyme on ice for 30 min,followed by sonication in the column buffer for 200 sec in periods of 10sec followed by a pause of 30 sec. After centrifugation at 9.000 g, thefusion proteins were purified from the crude extracts by affinitychromatography on amylose resin column. The MPB fusion protein binds toamylose. After extensive washes of the column, the fusion proteins wereeluted with 10 mM maltose.

Aliquots of the fractions were analyzed on 10% SDS-PAGE (Laemmli, U. K.1970. Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature 277, 680-685). Fractions containing the fusionprotein of interest were pooled and dialysed against 4×100 volumes ofPBS, pH 7.3, and the protein concentration was determined as describedby Bradford.

Southern blotting

Mycobacterial genomic DNA was prepared as described by Andersen et al.,1992. The Mycobacterium strains used in this study are listed in Table2.

M. leprae Armadillo-derived chromosomal DNA was obtained from M.Colston, Mill Hill, London, England.

Four μg of chromosomal DNA was digested with EcoRI, elctrophoresed in an0.8% agarose gel, and transferred onto Gene-Screen Plus membranes (NENResearch Products, Boston, Mass.). Hybridization was performed at 65° C.in an aqueous solution containing 1% SDS, 1M NaCl, 10% dextran sulfate,100 μg of denatured salmon sperm DNA per ml, and a (α-³² P) dCTPnick-translated MPT64 DNA probe.

The distribution of MPT64 in different M. tuberculosis substrains, M.bovis BCG substrains, and in M. leprae by the use of the 628 bp MPT64nucleotide fragment from pTO1 as a prove in Southern blot experiments isshown in FIG. 11. The probe hybridized to EcoRI fragments of app. 14 kbfor M. tuberculosis H37Rv, of app. 12 kb for M. tuberculosis H37Ra andM. tuberculosis Erdman, of app. 20 kb for M. bovis BCG Tokyo, and ofapp. 9.5 kb for M. bovis BCG Moreau and M. bovis BCG Russian, but theprobe did not hybridize to any EcoRI fragments from M. bovis BCG Glaxo,M. bovis BCG Pasteur, M. bovis BCG Canadian, M. bovis BCG Tice, M. bovisBCG strain; Danish 1331, and M.laprae.

                  TABLE 2                                                         ______________________________________                                        Mycobacterial strains used in this study                                      No.  Strain           Source                                                  ______________________________________                                        1    M. tuberculosis H37Rv                                                                          ATCC* No. 27294                                         2    M. tuberculosis Erdman                                                                         Obtained from A. Lazlo, Canada.                         3    M. tuberculosis H37Ra                                                                          ATCC* No. 25177                                         4    M. bovis Calmette Guerin                                                                       Copenhagen BCG Laboratory,                                                    SSI.sup.+                                               5    M. bovis Calmette Guerin                                                                       Tokyo.sup.#                                             6    M. bovis Calmette Guerin                                                                       Moreau.sup.#                                            7    M. bovis Calmette Guerin                                                                       Russian.sup.#                                           8    M. bovis Calmette Guerin                                                                       Glaxo.sup.#                                             9    M. bovis Calmette Guerin                                                                       Pasteur.sup.#                                           10   M. bovis Calmette Guerin                                                                       Canadian.sup.#                                          11   M. bovis Calmette Guerin                                                                       Tice.sup.#                                              12   M. leprae Armadillo-derived                                                                    Obtained from M J Colston,                                                    England                                                 ______________________________________                                         *American Type Culture Collection, USA.                                       .sup.+ Statens Seruminstitut, Denmark.                                        .sup.# WHO International Laboratory for Biological Standards, Statens         Seruminstitut, Denmark.                                                  

EXAMPLE 6

Skin testing of guinea pigs with rMPT64

The immunological potential of MPT64 was assessed by the followingexperiment:

A group of outbred guinea pigs (strain Ssc:Al) was immunized with eithM. bovis BCG Danish 1331 or with M. bovis BCG Tokyo. All guinea pigswere skin tested with 0.1 μg purified rMPT64 (recombinant MPT64), nativeMPT64, and 2 T.U. of tuberculin as described by Andersen et al. (1991)in Scand. J. Immun. 59: 365-372. The skin reactions to tuberculin wereused as a positive control of the ummunization. Skin reactions in theguinea pigs to the purified recombinant antigen is shown in FIG. 12.

rMPT64 elicited Dth reactions in the guinea pigs sensitized with M.bovis BVD Tokyo, the skin reactions are comparable to those obtained bypurified MPT64, no skin reactions were seen in guinea pigs sensitizedwith M. bovis BCG Danish 1331. In conclusion rMPT64 is as suitable aspurified MPT64 as a skin test reagent, and rMPT64 has, like MPT64, aspecificity superior to tuberculin preparations.

EXAMPLE 7

T-cell epitope mapping on rMPT64 fusion proteins by skin testing guineapigs

In order to map specific T-cell epitopes on rMPT64 fusion proteins byskin testing, a group of outbred guinea pigs (strain SSc:Al) wasimmunized with either M. bovis BCG Danish 1331 or M. bovis BCG Tokyo.The sensitized guinea pigs were skin tested with C- and N-terminallytruncated versions of rMPT64 as MBP fusion proteins (cf. example 4).

The fusion proteins were semi-purified by affinity chromatography on anamylose resin column followed by FPLC (Fast Performance LiquidChromatography) over an anion exchange column (Mono Q) as describedabove. Aliquots of the fractions were analyzed on 10% SDS-PAGE.Fractions containing semi-purified recombinant fusion proteins werepooled and dialysed extensively against physiological saline before use.The skin test experiments with the recombinant fusion proteins indicatedthat the specific T-cell epitope is located within the sequence encodedby TO40 (FIGS. 13 and 14).

In order to further confirm the localization of the T-cell epitope,similar experiments were performed using synthetic peptides derived fromthe carboxy terminal end of MPT64 instead of deletion mutants. The aminoacid sequences of the peptides employed can be seen in FIG. 15, and theresults of the skin tests can be seen from FIG. 16.

As can be seen from FIG. 16, the reactive synthetic fragments are C3,C4, and D1. These results confirm that the location of the T-cellepitope is found between the amino acids 186-215 in SEQ ID NO: 2.

EXAMPLE 8

Kinetics of the skin inducing capacity of MPT64 compared to PPD RT23 inaerosol infected guinea pigs

In order to establish how early in the course of infection guinea pigsare converted to MPT64-positives, the following experiment was carriedout:

Four groups of guinea pigs (n=5) were exposed to aerosols of M.tuberculosis Erdman at doses giving rise to an average of 5 primarytuberculous lesions per lung. Skin testings were performed after 3, 6,8, and 11 weeks after inhalation, cf. FIG. 17. All the animals tested 6weeks after exposure mounted a positive skin test reaction to MPT64.This result is in agreement with the conventional tuberculin skintestings, cf. FIG. 18.

The conclusion is that reactivity towards MPT64 has an onset atapproximately the same stage after infection with mycobacteria as theonset of the tuberculin reaction.

EXAMPLE 9

Comparison of specific, skin test inducing activity of native MPT64 withMPT64 molecules which have been chemically modified by pretreatment witheither glutaraldehyde or formaldehyde

It is contemplated that reactivity of MPT64 or analogues thereof will beenhanced if it is possible to maintain a high local concentration of theantigen at the injection site. Thereby is obtained that the intensity ofthe reaction is increased and the persistence at the injection siteprolonged. One possible strategy which is expected to have this resultis the use of homopolymers of MPT64 made by chemical treatment with thedenaturing agents glutaraldehyde or formaldehyde.

In order to test whether this strategy is likely to lead to the expectedresult, it is planned to perform the following experiment:

40 μg of native MPT64 is incubated at 37° C. for 8 days in the presenceof variouys concentrations of glutaraldehyde or formaldehyde (0.005M,0.025M, and 0.125M) in a total volume of 1.0 ml PBS. The samples aresubsequently dialysed against 100 ml of PBS four times. The proteinconcentration in the final sample preparation is then checked againbefore being injected into guinea pigs, which have been immunized witheither BCG Tokyl or BCG Danish 1331. The reactions should be read after24, 48, and 72 hours.

LIST OF REFERENCES

Andersen, Å. B., Andersen, P., Ljungqvist, L. Infection and Immunity 60:2317-2323, 1992.

Andersen, Å. B., Ljungqvist, L., Haslφv, K., Bentzon, M. W. Scand. J.Immunol. 34: 365-372, 1991.

Bullock, W. O., Fernandez, J. M., Short, J. M. Biotechniques 5: 376-379,1987.

Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. J. AcademicPress, 253-258, 1990.

Laemnli, U. K. Nature 277: 680-685, 1970.

Lotte, A., Wasz-Hockert, O., Poisson, N., Dumitrescu, N., Verron, M.Couvet, E. Adv. Tuberc. Res. 21: 107-193, 1984.

Maniatis, T., Fritsch, Sambrook, J. Molecular cloning. A LaboratoryManual. Second edition. Cold Spring Harbor Laboratory Press 1989

Nagai, S. Wiker, H. G., Harboe, M., Kinomoto, M. Infection and Immunity59: 372-382, 1991.

Worsaae, A., Ljungqvist, L., Haslφv, K., Heron, I., Bennedsen, J.Infection and Immunity 55: 2922-2927, 1987.

Yamaguchi, R. Matsuo, K., Yamazaki, A., Abe, C., Nagai, S., Terasaka, K.Yamada, T. Infection and Immunity 57: 283-288, 1989.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 11                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 826 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (vi) ORIGINAL SOURCE:                                                             (A) ORGANISM: Mycobacteriu - #m tuberculosis                                  (B) STRAIN: H37Rv                                                   -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 139..822                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: sig.sub.-- - #peptide                                           (B) LOCATION: 139..207                                              -     (ix) FEATURE:                                                           10.sub.-- - #signalKEY:                                                                 (B) LOCATION: 27..32                                                -     (ix) FEATURE:                                                           35.sub.-- - #signalKEY:                                                                 (B) LOCATION: 51..56                                                #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - TCTGCTAGCT TGAGTCTGGT CAGGCATCGT CGTCAGCAGC GCGATGCCCC TA - #TGTTTGTC         60                                                                          - GTCGACTCAG ATATCGCGGC AATCCAATCT CCCGCCTGCG CCGGCGGTGC TG - #CAAACTAC        120                                                                          #CTG GTC ACG GCT      171 CGC ATC AAG ATC TTC ATG                             #Thr Alarg Ile Lys Ile Phe Met Leu Val                                        # 10                                                                          - GTC GTT TTG CTC TGT TGT TCG GGT GTC GCC AC - #G GCC GCG CCC AAG ACC          219                                                                          Val Val Leu Leu Cys Cys Ser Gly Val Ala Th - #r Ala Ala Pro Lys Thr           #             25                                                              - TAC TGC GAG GAG TTG AAA GGC ACC GAT ACC GG - #C CAG GCG TGC CAG ATT          267                                                                          Tyr Cys Glu Glu Leu Lys Gly Thr Asp Thr Gl - #y Gln Ala Cys Gln Ile           #         40                                                                  - CAA ATG TCC GAC CCG GCC TAC AAC ATC AAC AT - #C AGC CTG CCC AGT TAC          315                                                                          Gln Met Ser Asp Pro Ala Tyr Asn Ile Asn Il - #e Ser Leu Pro Ser Tyr           #     55                                                                      - TAC CCC GAC CAG AAG TCG CTG GAA AAT TAC AT - #C GCC CAG ACG CGC GAC          363                                                                          Tyr Pro Asp Gln Lys Ser Leu Glu Asn Tyr Il - #e Ala Gln Thr Arg Asp           # 75                                                                          - AAG TTC CTC AGC GCG GCC ACA TCG TCC ACT CC - #A CGC GAA GCC CCC TAC          411                                                                          Lys Phe Leu Ser Ala Ala Thr Ser Ser Thr Pr - #o Arg Glu Ala Pro Tyr           #                 90                                                          - GAA TTG AAT ATC ACC TCG GCC ACA TAC CAG TC - #C GCG ATA CCA CCG CGT          459                                                                          Glu Leu Asn Ile Thr Ser Ala Thr Tyr Gln Se - #r Ala Ile Pro Pro Arg           #            105                                                              - GGT ACG CAG GCC GTG GTG CTC AAG GTC TAC CA - #G AAC GCC GGC GGC ACG          507                                                                          Gly Thr Gln Ala Val Val Leu Lys Val Tyr Gl - #n Asn Ala Gly Gly Thr           #       120                                                                   - CAC CCA ACG ACC ACG TAC AAG GCC TTC GAT TG - #G GAC CAG GCC TAT CGC          555                                                                          His Pro Thr Thr Thr Tyr Lys Ala Phe Asp Tr - #p Asp Gln Ala Tyr Arg           #   135                                                                       - AAG CCA ATC ACC TAT GAC ACG CTG TGG CAG GC - #T GAC ACC GAT CCG CTG          603                                                                          Lys Pro Ile Thr Tyr Asp Thr Leu Trp Gln Al - #a Asp Thr Asp Pro Leu           140                 1 - #45                 1 - #50                 1 -       #55                                                                           - CCA GTC GTC TTC CCC ATT GTG CAA GGT GAA CT - #G AGC AAG CAG ACC GGA          651                                                                          Pro Val Val Phe Pro Ile Val Gln Gly Glu Le - #u Ser Lys Gln Thr Gly           #               170                                                           - CAA CAG GTA TCG ATA GCG CCG AAT GCC GGC TT - #G GAC CCG GTG AAT TAT          699                                                                          Gln Gln Val Ser Ile Ala Pro Asn Ala Gly Le - #u Asp Pro Val Asn Tyr           #           185                                                               - CAG AAC TTC GCA GTC ACG AAC GAC GGG GTG AT - #T TTC TTC TTC AAC CCG          747                                                                          Gln Asn Phe Ala Val Thr Asn Asp Gly Val Il - #e Phe Phe Phe Asn Pro           #       200                                                                   - GGG GAG TTG CTG CCC GAA GCA GCC GGC CCA AC - #C CAG GTA TTG GTC CCA          795                                                                          Gly Glu Leu Leu Pro Glu Ala Ala Gly Pro Th - #r Gln Val Leu Val Pro           #   215                                                                       #         826      AC TCG ATG CTG GCC TAGA                                    Arg Ser Ala Ile Asp Ser Met Leu Ala                                           220                 2 - #25                                                   - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 228 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Val Arg Ile Lys Ile Phe Met Leu Val Thr Al - #a Val Val Leu Leu Cys         #                 15                                                          - Cys Ser Gly Val Ala Thr Ala Ala Pro Lys Th - #r Tyr Cys Glu Glu Leu         #             30                                                              - Lys Gly Thr Asp Thr Gly Gln Ala Cys Gln Il - #e Gln Met Ser Asp Pro         #         45                                                                  - Ala Tyr Asn Ile Asn Ile Ser Leu Pro Ser Ty - #r Tyr Pro Asp Gln Lys         #     60                                                                      - Ser Leu Glu Asn Tyr Ile Ala Gln Thr Arg As - #p Lys Phe Leu Ser Ala         # 80                                                                          - Ala Thr Ser Ser Thr Pro Arg Glu Ala Pro Ty - #r Glu Leu Asn Ile Thr         #                 95                                                          - Ser Ala Thr Tyr Gln Ser Ala Ile Pro Pro Ar - #g Gly Thr Gln Ala Val         #           110                                                               - Val Leu Lys Val Tyr Gln Asn Ala Gly Gly Th - #r His Pro Thr Thr Thr         #       125                                                                   - Tyr Lys Ala Phe Asp Trp Asp Gln Ala Tyr Ar - #g Lys Pro Ile Thr Tyr         #   140                                                                       - Asp Thr Leu Trp Gln Ala Asp Thr Asp Pro Le - #u Pro Val Val Phe Pro         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ile Val Gln Gly Glu Leu Ser Lys Gln Thr Gl - #y Gln Gln Val Ser Ile         #               175                                                           - Ala Pro Asn Ala Gly Leu Asp Pro Val Asn Ty - #r Gln Asn Phe Ala Val         #           190                                                               - Thr Asn Asp Gly Val Ile Phe Phe Phe Asn Pr - #o Gly Glu Leu Leu Pro         #       205                                                                   - Glu Ala Ala Gly Pro Thr Gln Val Leu Val Pr - #o Arg Ser Ala Ile Asp         #   220                                                                       - Ser Met Leu Ala                                                             225                                                                           - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24CCTA CTGC                                                  - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             27   TTAC ATCGCCC                                               - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             27   CTAC CAGAACG                                               - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             27   CTAT CGCAAGC                                               - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             27   GCAG ACCGGAC                                               - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #             27   GGTG AATTATC                                               - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #21                TTGA G                                                     - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -    (iii) ANTI-SENSE: YES                                                    #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                24TCGA GTCG                                                  - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -    (iii) ANTI-SENSE: YES                                                    #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   #                23GTAG ACC                                                   __________________________________________________________________________

What is claimed is:
 1. A kit comprising:(A) a BCG vaccine againsttuberculosis, and (B) at least one diagnostic test comprising apharmaceutical composition comprising a polypeptide, wherein:(i) thepolypeptide has a length of at least 12 amino acid residues, and thepolypeptide has a sequence comprising at least 12 amino acid residuesfrom a polypeptide of a mycobacterium of the tuberculosis complex(Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacteriumbovis), and (ii) lymphoid cells previously primed with virulentmycobacteria belonging to the tuberculosis complex (Mycobacteriumtuberculosis, Mycobacterium africanum and Mycobacterium bovis) arecapable of reacting with the polypeptide and (iii) lymphoid cellspreviously primed with a vaccine against tuberculosis are not capable ofreacting with the polypeptide, and (iv) a positive response isindicative of having or having had tuberculosis, and (v) a negativeresponse is indicative of not having or not having had tuberculosis. 2.A kit according to claim 1, wherein the pharmaceutical compositioncomprises a homopolymer of the polypeptide.
 3. A kit according to claim2, wherein the homopolymer is produced by introduction of at least onecyteine residue in the N-terminal region of the polypeptide.
 4. A kitaccording to claim 1, wherein the pharmaceutical composition comprises aheteropolymer of the polypeptide.
 5. A kit according to claim 4, whereinthe heteropolymer comprises the polypeptide coupled to a carrier orvehicle.
 6. A kit according to claim 4, wherein the heteropolymercomprises the polypeptide coupled to another mycobacterial protein.
 7. Akit according to claim 1, wherein the polypeptide has beenpost-translationally modified.
 8. A kit according to claim 1, whereinthe positive response is a positive skin response which appears 1-4 daysafter injection.
 9. A kit according to claim 1, wherein the positiveresponse is a positive skin response between 0.5 cm and 4.0 cm indiameter.
 10. A kit acording to claim 1, wherein the polypeptide has anamino acid sequence comprising the amino acid sequence SEQ ID NO:
 2. 11.A kit according to claim 1, wherein the polypeptide is encoded by a DNAmolecule having a nucleotide sequence comprising the nucleotide sequenceSEQ. ID NO:
 1. 12. A kit according to claim 1, wherein the polypeptidehas been modified in order to abolish or delete sensitizing epitopes.13. A kit according to claim 1, wherein the polypeptide has beenmodified by denaturing procedures.
 14. A kit according to claim 1,wherein the polypeptide comprises MPT64.
 15. A kit according to claim 1,wherein the pharmaceutical composition comprises at least two differentpolypeptides.
 16. A ket according to claim 15 comprising a firstpolypeptide comprising MPT64 and a second polypeptide comprising MPT59.17. A kit according to claim 14, wherein the pharmaceutical compositioncomprises 0.05 to 20 μg of the polypeptide.
 18. A kit comprising:(A) aBCG vaccine against tuberculosis, wherein said BCG vaccine does notcontain MPT64 or MPT59 or a fragment thereof having a length of at least12 amino acids from MPT64 or MPT59, and (B) at least one diagnostic testcomprising a pharmaceutical composition comprising a polypeptide,wherein:(i) the polypeptide comprises MPT64, or MPT59, or MPT64 andMPT59, or a fragment of MPT64 or MPT59, or a fragment of MPT64 andMPT59; said fragment having a length of at least 12 amino acid residuesfrom MPT64 and/or MPT59, and (ii) lymphoid cells previously primed withvirulent mycobacteria belonging to the tuberculosis complex(Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacteriumbovis) are capable of reacting with the polypeptide and (iii) lymphoidcells previously primed with a vaccine against tuberculosis are notcapable of reacting with the polypeptide, and (iv) a positive responseis indicative of having or having had tuberculosis, and (v) a negativeresponse is indicative of not having or not having had tuberculosis. 19.A method of diagnosing tuberculosis caused by Mycobacterium tuberculosi,Mycobacterium africanum or Mycobacterium bovis in an individualsusceptible thereto, comprising:exposing immune cells of the individualto a pharmaceutical composition comprising a polypeptide:having a lengthof at least 12 amino acid residues, wherein the polypeptide has asequence comprising at least 12 amino acid resiudes from a polypeptideof a mycobacterium of the tuberculosis complex (Mycobacteriumtuberculosis, Mycobacterium africanum and Mycobacterium bovis), and withwhich lymphoid cells previously primed with virulent mycobacteriabelonging to the tuberculosis complex (Mycobacterium tuberculosis,Mycobacterium africanum and Mycobacterium bovis) are capable of reactingand with which lymphoid cells previously primed with a BCG vaccineagainst tuberculosis are not capable of reacting,wherein a positiveresponse is indicative of the individual having or having hadtuberculosis, and a negative response is indicative of the individualnot having or not having had tuberculosis.
 20. A method of diagnosingtuberculosis caused by Mycobacterium tuberculosis, Mycobacteriumafricanum or Mycobacterium bovis in an individual susceptible thereto,comprising:exposing immune cells of the individual to a pharmaceuticalcomposition comprising a polypeptide:having a length of at least 12amino acid residues, wherein the polypeptide has a sequence comprisingat least 12 amino acid residues from a polypeptide of a mycobacterium ofthe tuberculosis complex (Mycobacterium tuberculosis, Mycobacteriumafricanum and Mycobacterium bovis), and with which lymphoid cellspreviously primed with virulent mycobacteria belonging to thetuberculosis complex (Mycobacterium tuberculosis, Mycobacteriumafricanum and Mycobacterium bovis) are capable of reacting and withwhich lymphoid cells previously primed with a BCG vaccine againsttuberculosis are not capable of reacting,wherein a positive response isindicative of the individual having or having had tuberculosis, and anegative response is indicative of the individual not having or nothaving had tuberculosis, and the polypeptide is as in the dit of claim2-7, 10,11-13, 14-16, 17, or
 18. 21. A method of diagnosing tuberculosiscaused by Mycobacterium tuberculosis, Mycobacterium africanum orMycobacterium bovis in an individual susceptible thereto,comprising:exposing immune cells of the individual to a pharmaceuticalcomposition comprisinga polypeptide comprising MPT64, or MPT59, or MPT64and MPT59, or a fragment of MPT64 or MPT59 or a fragment of MPT64 andMPT59; said fragment having a length of at least 12 amino acid residuesfrom MPT64 and/or MPT59, and with which lymphoid cells previously primedwith virulent mycobacteria belonging to the tuberculosis complex(Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacteriumbovis) are capable of reacting and with which lymphoid cells previouslyprimed with a BCG vaccine against tuberculosis are not capable ofreacting,wherein the BCG vaccine does not contain MPT64 or MPT59 or afragment thereof having a length of at least 12 amino acids from MPT64or MPT59, a positive response is indicative of the individual having orhaving had tuberculosis, and a negative response is indicative of theindividual not having or not having had tuberculosis.
 22. The method ofclaim 19 wherein the pharmaceutical composition comprises a homopolymerand/or a heteropolymer of the polypeptide.
 23. The method of claim 19wherein the polypeptide comprises: MPT64; or MPT59; or a polypeptideencoded by a nucleic acid molecule comprising MPT64-4, MPT64-5, MPT64-6,MPT64-7, MPT64-8; or a polypeptide comprising amino acids 186-215 of SEQID NO:2; or a polypeptide comprising an amino acid sequence as set forthin SEQ ID NO:2; or a polypeptide comprising a polypeptide encoded by thesequence set forth in SEQ ID NO:1.
 24. An isolated polypeptideconsisting of an amino acid sequence as set forth in SEQ ID NO:2. 25.The isolated polypeptide according to claim 24, which has been modifiedin order to abolish or delete sensitizing epitopes.
 26. The isolatedpolypeptide according to claim 24 containing at least one T-cellepitope.
 27. An isolated polypeptide consisting of the amino acidsequence 186-215 in SEQ ID NO:2.
 28. The kit of claim 1 wherein thepolypeptide comprises a polypeptide encoded by a nucleic acid moleculecomprising MPT64-4, MPT64-5, MPT64-6, MPT64-7, MPT64-8; or thepolypeptide comprises amino acids 186-215 of SEQ ID NO:2.
 29. A methodof producing an isolated polypeptide as defined in claim 24, the methodcomprising sequentially linking component amino acids to form thepolypeptide.
 30. A kit according to claim 2, wherein the homopolymercomprises at least two copies of the peptide.
 31. A kit according toclaim 7, wherein the polypeptide has been post-translationally acylated,post-translationally glycosylated, or post translationally acylated andglycosylated.
 32. A kit according to claim 8, wherein the positive skinresponse appears 2-3 days after injection.
 33. A kit according to claim9, wherein the positive skin response is between 1.0 cm and 3.0 cm indiameter.
 34. A kit according to claim 18, wherein the polypeptidecomprises MPT64 or MPT59.
 35. A kit according to claim 13, wherein thedenaturing procedures are selected from the group consisting ofautoclaving, and treatment with formaldelyde or glutaraldehyde.
 36. Akit according to claim 17 wherein the pharmaceutical compositioncomprises 0.5 to 2.0 μg of the polypeptide.
 37. A kit according to claim17 wherein the pharmaceutical composition comprises 0.75 to 1.5 μg ofthe polypeptide.
 38. The method of claim 29 of producing an isolatedpolypeptide comprising solid or liquid phase peptide synthesis.
 39. Akit comprising:(A) a BCG vaccine against tuberculosis comprising anamount of a BCG vaccine against tuberculosis, and (B) at least onediagnostic test comprising a pharmaceutical composition comprising apolypeptide,wherein (i) the polypeptide comprises a polypeptide encodedby a DNA molecule having a nucleotide sequence comprising the nucleotidesequence SEQ ID NO: 1 and (ii) lymphoid cells previously primed withvirulent mycobacteria belonging to the tuberculosis complex(Mycobacterium tuberculosis, Mycobacterium africanum and Mycobacteriumbovis) are capable of reacting with the polypeptide and (iii) lymphoidcells previously primed with the vaccine are not capable of reactingwith the polypeptide, and (iv) intradermal injection of the polypeptideat a location of injection elicits a skin reaction having a diameter ofat least 0.5 cm in an individual previously sensitized with virulentmycobacteria belonging to the tuberculosis complex and, (v) a positiveresponse is indicative of the person having or having had tuberculosis,and (vi) a negative response is indicative of the person not having ornot having had tuberculosis.
 40. The kit of claim 1 wherein thediagnostic test is a diagnostic skin test for intradermal injection, thepositive response is a positive response at the location of injection,and the negative response is a negative response at the location ofinjection.
 41. The kit of claim 18 wherein the BCG vaccine is BCGstrain: Danish
 1331. 42. The method of claim 19 wherein exposing immunecells is by intradermal injection, the positive response is a positiveskin response at the location of injection, and the negative response isa negative skin response at the location of injection.
 43. The method ofclaim 21 wherein the BCG vaccine is BCG strain: Danish
 1331. 44. Themethod of claim 31 wherein the polypeptide is from a mycobacteriumbelonging to the tuberculosis complex.
 45. The method according to claim20, wherein the BCG vaccine is from BCG strain: Danish
 1331. 46. Themethod according to claim 20, wherein the polypeptide comprises apolypeptide encoded by a nucleic acid molecule comprising MPT64-4,MPT64-5, MPT64-6, MPT64-7, MPT64-8; or the polypeptide comprises aminoacids 186-215 of SEQ ID NO:2.
 47. The kit of claim 39 wherein thediagnostic test is a diagnostic skin test for intradermal injection, thepositive response is a positive response at the location of injection,and the negative response is a negative response at the location ofinjection.
 48. The method of claim 20 wherein exposing immune cells isby intradermal injection, the positive response is a positive skinresponse at the location of injection, and the negarive response is anegative skin response at the location of injection.
 49. The kit ofclaim 1 wherein the polypeptide comprises a polypeptide from amycobacterium of the tuberculosis complex (Mycobacterium tuberculosis,Mycobacterium africanum and Mycobacterium bovis).
 50. The methodaccording to claim 19 wherein the polypeptide comprises a polypeptidefrom a mycobacterium of the tuberculosis complex (Mycobacteriumtuberculosis, Mycobacterium africanum and Mycobacterium bovis).